CFTR polypeptides, fragments thereof and methods of use to overcome biosynthetic misprocessing

ABSTRACT

The invention relates to biosynthetic maturation of cell surface polypeptides and, more specifically, to particular CFTR polypeptides which exhibit increased transport to the cell surface and tripeptide amino acid sequences that promote or enhance transport of export-incompetent CFTR to the cell surface.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support awarded by the NationalInstitutes of Health. The Government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

The invention relates to biosynthetic maturation of cell surfacepolypeptides and, more specifically, to particular CFTR polypeptideswhich exhibit increased transport to the cell surface and tripeptideamino acid sequences that promote or enhance transport ofexport-incompetent CFTR to the cell surface.

Large multidomain and multisubunit proteins are assembled into theirnative tertiary and quaternary structures, respectively, in theendoplasmic reticulum (ER). If this assembly is imperfect because ofmutations or for other reasons, the aberrant protein is targeted fordegradation by a set of processes generally referred to as biosyntheticquality control. Although a great deal has been learned about theseprocesses in the past few years the rate-limiting step determiningwhether or not a nascent chain is exported from the ER has not beenidentified. Indeed, the relation of the folding and degradationmechanisms to the ER export and retrieval pathways is not understood forany cell surface or secreted molecule.

Cystic fibrosis (CF) is an example of a disease which may benefit froman understanding of the factors that contribute to or mediate retentionor export of proteins from the endoplasmic reticulum because, in manypatients, ER-retention prevents potentially functional variant CFTR(Cystic Fibrosis Transmembrane Conductance Regulator) molecules fromreaching the plasma membranes of secreting and reabsorbing epithelialcells, where CFTR is required as a regulated chloride channel. Forexample, although more than 800 different mutations in the CFTR genehave been detected in CF patients, more than 90% of patients possess asingle misprocessing mutant, ΔF508. Collins, F. S. (1992) Science256:774–779. This mutant protein is potentially functional as aregulated chloride channel if it can be made to move further along thesecretory pathway and reach the cell surface. Dalemans, W. et al. (1991)Nature 354:526–528; Drumm, M. L. et al. (1991). Science 254:1797–1799;and Li, C. et al. (1993) Nat. Genetics 3:311–316.

Ubiquitination ultimately marks maturation-incompetent nascent chains assubstrates for the proteasome but earlier steps in the recognition ofthese targets are unclear. Jensen, T. J. et al. (1995) Cell 83:129–135;Sato, S. et al. (1998). J. Biol. Chem. 273:7189–92; and Ward, C. L etal. (1995) Cell 83:121–127. Molecular chaperones can either retainunfolded proteins or assist in their folding. Nascent CFTR interactswith chaperones on both sides of the ER membrane; on the cytoplasmicface of the ER, Hsp70 (Yang, Y. et al. (1993) Proc. Natl. Acad. Sci. USA90:9480–9484) and its cochaperone, Hdj-2 (Meacham, G. C. et al. (1999)EMBO J. 18:1492–1505) as well as Hsp90 (Loo et al. (1998) EMBO J.17:6879–6887) bind to immature CFTR. Other chaperones could also bepresent in the large multimolecular complexes containing nascent CFTR.Pind, S. et al. (1994) J. Biol. Chem. 269:12784–12788. Although theseinteractions appear to occur with both wild-type CFTR and mutant ΔF508CFTR and no large differences in the kinetics or stoichiometry ofCFTR-chaperone interactions have yet been found, the role of chaperonesin ER retention of CFTR cannot be ruled out.

In addition to chaperones, short sequence motifs have been shown toexert positive and negative effects on secretory proteins which arerequired for ER export and retrieval, respectively. For example, a shortdiacidic ER export signal has been described as necessary for transportof VSV-G glycoprotein from the ER. Nishimura, N., and Balch, W. E.(1997) Science 277:556–8. Whether nascent ΔF508 CFTR never leaves the ERor if it is retrieved is not known, although ΔF508 CFTR may reach theintermediate compartment (ERGIC) between ER and Golgi. Gilbert, A. etal. (1998) Exp. Cell Res. 242:144–52.

Previous attempts to overcome ER-retention of mutant CFTR have includedinhibition of the proteasome involved in nascent chain proteolysis(Jensen et al., 1995, supra; Ward et al., 1995, supra), perturbation ofinteraction with molecular chaperons (Jiang, C. et al. (1998). Am. J.Physiol. 275:C 171–8; and Loo et al., 1998, supra) and using agents orconditions which influence protein folding for example, glycerol andother osmolytes (Brown, C. R. et al. (1996) Cell Stress Chaperones1:117–25; Qu, B. H. et al. (1997) J. Biol. Chem. 272:15739–44; and Sato,S. et al. (1996) J. Biol. Chem. 271:635–638) or reduced temperature(Denning, G. M. et al. (1992) Nature 358:761–764). However, thesetreatments are minimally effective or extremely toxic to cells,precluding their application to patients.

Thus, there remains a need to understand the biological basis for ERretention, especially in respect to the retention of proteins such asCFTR that have severe physiological consequences. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that anexport-incompetent CFTR has several arginine-framed tripeptide sequencemotifs that contribute to the observed deficiency of cell surfaceexpression, and that modifying one or more of these sequences can resultin increased transport of the export-incompetent CFTR to the cellsurface.

In one aspect, the invention features an isolated polypeptide thatincludes a polypeptide having the sequence X1-R-X2-R-X3, wherein X1, X2and X3 are any amino acid and polynucleotides encoding suchpolypeptides. The polypeptide can have at least seven amino acids. Theinvention also features an isolated polypeptide selected from the groupconsisting of: GYRQRLE (SEQ ID NO:1), EYRYRSV (SEQ ID NO:2), GQRARIS(SEQ ID NO:3) and QARRRQS (SEQ ID NO:4), and polynucleotides encodingsuch polypeptides. Polypeptides of the invention further can include apharmaceutical formulation.

The invention also features an isolated polypeptide having one or moreR-X-R sequences, wherein at least one R of an R-X-R sequence has beensubstituted with another amino acid, and wherein the substitutedpolypeptide is exported from the ER in an amount or at a rate greaterthan the unsubstituted polypeptide. The polypeptide can be CFTR.

In another aspect, the invention features a method for identifying atherapeutic agent for treating cystic fibrosis. The method includescontacting at least one cell expressing an export-incompetent CFTR witha test agent under conditions allowing an interaction between the agentand a factor mediating or contributing to export-incompetence; anddetermining whether the agent increases the amount of the CFTR on thecell surface, where an increased amount of CFTR on the cell surfaceidentifies a therapeutic agent for treating cystic fibrosis.

In yet another aspect, the invention features a method for identifyingan agent that induces or increases transport of an export-incompetentprotein. The method includes contacting an export-incompetent proteinwith a test agent under conditions allowing an interaction between theagent and a factor mediating or contributing to export-incompetence; anddetermining whether the agent increases the amount or the rate ofprotein transported, where an increased amount of transported proteinidentifies an agent that induces or increases transport of anexport-incompetent protein. The protein can be a secreted protein, acell surface protein, or CFTR. The method can be performed in vitro orthe export-incompetent protein can be expressed in a cell.

The invention also features a method for treating a subject havingcystic fibrosis. The method includes administering to the subject apharmaceutical formulation comprising a polypeptide having an R-X-Rsequence in an amount effective for treating cystic fibrosis. In oneembodiment, the X amino acid of the R-X-R sequence is not alanine,asparagine or glutamate. The polypeptide can have at least four aminoacids, e.g., at least seven amino acids. The polypeptide can be selectedfrom the group consisting of: GYRQRLE (SEQ ID NO:1), EYRYRSV (SEQ IDNO:2), GQRARIS (SEQ ID NO:3) and QARRRQS (SEQ ID NO:4).

In yet another aspect, the invention features a method for treating asubject having cystic fibrosis. The method includes administering to thesubject a pharmaceutical formulation that includes a nucleic acidencoding a polypeptide having an R-X-R sequence in an amount effectivefor treating cystic fibrosis.

In another aspect, the invention features a method for treating asubject having or suspected of having a physiological disorderassociated with an export-incompetent protein. The method includesadministering to the subject a pharmaceutical formulation comprising apolypeptide having an R-X-R sequence in an amount effective for treatinga physiological disorder associated with an export-incompetent protein.The physiological disorder or condition can be selected from the groupconsisting of: macular dystrophy and Stargardt's disease. Theexport-incompetent protein can be selected from the group consisting of:ion channels, ABC proteins, growth factors, immune regulators, adhesionproteins, hormones, clotting factors, hemostatic regulators andreceptors thereof. In one embodiment, the X amino acid of the R-X-Rsequence is not alanine, asparagine or glutamate. The polypeptide canhave at least four amino acids, e.g., at least seven amino acids. Thepolypeptide can be selected from the group consisting of: GYRQRLE (SEQID NO:1), EYRYRSV (SEQ ID NO:2, GQRARIS (SEQ ID NO:3) and QARRRQS (SEQID NO:4).

In yet another aspect the invention features a method for inducing orincreasing intracellular transport of an export-incompetent protein. Themethod includes contacting a cell expressing an export-incompetentprotein with a composition comprising a polypeptide having an R-X-Rsequence in an amount sufficient for inducing or enhancing intracellulartransport of the export-incompetent protein. The protein can have anR-X-R sequence and can be a cell surface protein, an export-incompetentCFTR, or a secreted protein.

A method for identifying an agent that inhibits or disrupts aninteraction between an R-X-R polypeptide and an ER retention factor alsois described. The method includes incubating a polypeptide having anR-X-R sequence and an ER retention factor under conditions allowingtheir interaction; adding a test agent to the incubation; and detectingbinding between the polypeptide and the ER retention factor, wheredecreased binding in the presence of the test agent identifies an agentthat inhibits or disrupts an interaction between an R-X-R polypeptideand an ER retention factor. The method can be performed in vitro. Thepolypeptide can have at least 4 amino acids. The X amino acid of theR-X-R sequence may not be alanine, asparagine or glutamate. Thepolypeptide can be selected from the group consisting of: GYRQRLE (SEQID NO:1), EYRYRSV (SEQ ID NO:2), GQRARIS (SEQ ID NO:3) and QARRRQS (SEQID NO:4).

The invention also features a method for identifying an ER retentionfactor. The method includes contacting a composition suspected ofcontaining an ER retention factor with a polypeptide having an R-X-Rsequence under conditions allowing interaction between the factor andthe polypeptide; and detecting binding between the factor and thepolypeptide, thereby identifying an ER retention factor. The method canbe performed in vitro. The polypeptide can have at least four aminoacids, e.g., at least seven amino acids. The polypeptide can be selectedfrom the group consisting of: GYRQRLE (SEQ ID NO:1), EYRYRSV (SEQ IDNO:2), GQRARIS (SEQ ID NQ:3) and QARRRQS (SEQ ID NO:4).

In yet another aspect, the invention features a method for inhibitingdegradation of a protein in a cell. The method includes contacting acell with a polypeptide having an R-X-R sequence in an amount sufficientfor inhibiting degradation of a cell surface or secreted protein. Theprotein can be a cell surface protein or a secreted protein.

A method for detecting the presence of an export-incompetent protein ina cell is also featured. The method includes contacting a cell with apolypeptide having an R-X-R sequence and detecting the intracellulartransport of the protein. The intracellular transport can be detectedusing an enzyme, by detecting the presence of the protein on the cellsurface, or by detecting secretion of the protein. The protein can beCFTR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1D show A) a schematic depiction of CFTR protein indicatingapproximate locations of arginine-framed tripeptide sequences andphenylalanine 508; B) western blots of wild-type and ΔF508 CFTRexpressed in BHK cells having the indicated substitutions; C) wild-typeand ΔF508 CFTR expressed in BHK cells having all four argininessubstituted with lysine (ΔF508/4RK) digested with endoglycosidase H; andD) surface biotinylation of mature CFTR on cells expressing ΔF508,ΔF508/4RK and wild-type CFTR. The arrowhead indicates position of matureband with complex (endoH resistant) oligosaccharide chains. Solid arrowsindicate the position of core-glycosylated band before endo-H digestionand open arrows are after endo-H digestion. Amino acid numbering beginsfrom the first methionine in CFTR. Expression, extraction, digestion andcell surface labeling was performed as described in Example I.

FIGS. 2A to 2D show the maturation of A) wild-type CFTR; B) wild-typeCFTR with arginine to lysine substitutions at all four arginine-framedtripeptides (wild-type/4RK; C) ΔF508 CFTR and; D) ΔF508 CFTR witharginine to lysine substitutions at all four arginine-framed tripeptides(ΔF508/4RK). Cells were pulse-labeled for 20 min and the chase times inhours are indicated. Maturation was assessed by the acquisition ofendoglycosidase H resistant oligosaccharide chains, which results in adecrease in the corresponding proteins mobility on SDS-PAGE.

FIGS. 3A to 3D show the localization of A) wild-type CFTR; B) wild-typeCFTR with arginine to lysine substitutions at all four arginine-framedtripeptides; C) ΔF508 CFTR and; D) ΔF508 CFTR with arginine to lysinesubstitutions at all four arginine-framed tripeptides, as determined byindirect immunofluorescence.

FIGS. 4A to 4D show the effect of AFT-containing heptamers on cellsexpressing wild-type CFTR-GFP fusion protein and ΔF508 CFTR-GFP fusionprotein. A) cells expressing wild-type CFTR-GFP fusion protein, notexposed to AFT-containing heptamer peptides; B) cells expressingwild-type CFTR-GFP fusion protein, exposed to AFT-containing heptamerpeptides; C) cells expressing ΔF508 CFTR-GFP fusion protein, not exposedto AFT-containing heptamer peptides; D) cells expressing ΔF508 CFTR-GFPfusion protein, exposed to AFT-containing heptamer peptides.

FIGS. 5A and 5B show the effect of the arginine to lysine substitutionson CFTR chloride channel activity from A) cells expressing wild-type andΔF508 CFTR with or without AFT mutations (³⁶ Cl⁻ efflux in cpm/min) andB) single CFTR Cl⁻ channels in planar lipid bilayers isolated from cellsexpressing wild-type, wild-type/4RK and ΔF508/4RK CFTR. The studies inA) were performed in triplicate, samples were averaged and the verticalbars represent standard deviation. In B), the arrows indicatestimulation with Forskolin and all points histograms are shown to theleft of the tracings.

FIG. 6 is an amino acid sequence of human cystic fibrosis transmembraneconductance regulator (CFTR) (SEQ ID NO:5).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery that particular mutantCFTR's are unable to be transported to the cell surface due, in part tothe presence of arginine-framed tripeptide sequence motifs, R-X-R,present in CFTR. There are four arginine-framed tripeptide sequencemotifs on CFTR that appear to mediate or contribute to retention ofmutant export-incompetent CFTR in the endoplasmic reticulum (ER),delaying or preventing transport of CFTR to the cell surface. Whenaltered, such as by substituting one arginine of the tripeptide sequencemotif with another amino acid, an increase in transport of thesubstituted CFTR to the cell surface in an amount greater thanunsubstituted export-incompetent CFTR is observed. Thus, agents thatinduce or increase transport of export-incompetent CFTR or otherexport-incompetent proteins from the ER can be used as therapeutics fortreating physiological diseases associated with export-incompetentproteins, such as cystic fibrosis (CF).

In one embodiment, the invention provides isolated CFTR polypeptideshaving one or more amino acid substitutions of an arginine-framedtripeptide (AFT). The substituted CFTR polypeptides are capable of beingtransported to the cell surface in an amount greater than anunsubstituted CFTR polypeptide. Thus, substituted CFTR polypeptides areuseful for the treatment of CF, for example. Moreover, as AFTs canmediate or contribute to a protein's inability to be transported fromthe ER, AFT and peptide sequences containing such AFT sequences areuseful as competitive inhibitors of ER retention factors that interactwith AFT containing polypeptides. As disclosed herein, competitiveinhibition using such peptides induces or enhances ER transport ofproteins, including export-incompetent proteins containing anarginine-framed tripeptide sequence. Thus arginine-framed tripeptidesare useful for treating physiological disorders related toexport-incompetent proteins, including cell surface as well as secretedproteins, such as CFTR. Thus, in another embodiment, the inventionprovides peptides having one or more arginine-framed tripeptidesequences, and methods of use.

As used herein, the terms “protein,” “polypeptide” and “peptide” areused interchangeably to denote an amino acid polymer that comprises atleast two amino acids covalently linked by an amide bond, regardless oflength or post-translational modification (e.g., glycosylation,phosphorylation, lipidation, ubiquitination etc.). D- and L-amino acids,and mixtures thereof are included.

As used herein, the terms “isolated” or “substantially pure,” when usedas a modifier of invention CFTR polypeptides, fragments thereof,arginine-framed tripeptides and nucleic acids, means that they areproduced by the hand of man and are separated from their native in vivocellular environment. Generally, polypeptides and nucleic acids soseparated are substantially free of other proteins, nucleic acids,lipids, carbohydrates or other materials with which they are naturallyassociated.

Typically, a polypeptide is substantially pure when it is at leas 60%,by weight, free from the proteins and naturally-occurring molecules withwhich it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably 95%, byweight. Substantially pure CFTR polypeptide can be obtained, forexample, by extraction from a natural source (e.g., an animal cell); byexpression of a recombinant nucleic acid encoding a CFTR polypeptide: orby chemically synthesizing the protein. Purity can be measured by anyappropriate method, e.g., polyacrylamide gel electrophoresis, or by HPLCanalysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants which accompany it in itsnatural state. Thus, a protein which is chemically synthesized orproduced in a cellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptidesinclude those derived from eukaryotic organisms but synthesized in E.coli or other prokaryotes.

As disclosed herein, substitution of one or more amino acids of an AFTsequence with a different amino acid can increase transport of anexport-incompetent polypeptide (see for example, Examples I to III).Thus, in another embodiment, the invention provides an isolatedpolypeptide containing at least one arginine-framed tripeptide. R-X-R,in which at least one R of the R-X-R tripeptide is substituted with anamino acid, and the substituted polypeptide is exported from theendoplasmic reticulum in an amount greater than the unsubstitutedpolypeptide. In one aspect, the first or third R is substituted or the Xposition amino acid is substituted. In another aspect, the first orthird R is substituted with a lysine. The X position amino acid is notsubstituted with alanine, asparagine or glutamate. Particular examplesof isolated polypeptides so substituted include export incompetent ΔF508CFTR, for example, R29K and R516K, in which the first arginine has beensubstituted with a lysine, R555K and R766K, in which the third argininehas been substituted with a lysine, and 4RK, in which all foursubstitutions are combined (FIG. 1B). Additional modifications of theCFTR sequence may influence ER-retention or export. For example,substitution of transport signals within the CFTR C-terminal extensionresidues (amino acid residues 1380–1480) also can increase ER export ofan export-incompetent CFTR.

As used herein the term “substituted,” when used as a modifier of apolypeptide containing at least one arginine-framed tripeptide (R-X-R),means that one or more amino acids of the R-X-R sequence or a sequencethat flanks R-X-R, has been substituted with another amino acid.Preferred amino acid substitutions occur at the first or third argininesof the R-X-R motif. More preferred substitutions occur at the firstarginine of the R-X-R motif. Generally, substitutions of the sequenceflanking R-X-R will be within about 15 amino acids of the motif,preferably within 10 amino acids and more preferably within five aminoacids. Multiple amino acid substitutions of R-X-R or a sequence thatflanks R-X-R also are included.

As used herein, the terms “export-incompetent” or “export-incompetence,”when used as a modifier of a protein, polypeptide or peptide; refers toa decrease or absence of intracellular transport from the endoplasmicreticulum (ER) to and through the golgi apparatus (cis, medial, trans)to an ultimate destination, such as the cell surface, to the lysosomesor endosomes, or secreted from the cell. A decrease either can occur inthe rate of transport (e.g., a time delay in the appearance of a proteinon the cell surface) or in the overall amount of protein transported toits ultimate destination, or a combination thereof. As disclosed herein,the rate or amount of intracellular transport of a protein can bedetermined by detecting the amount of protein present at the cellsurface or present in organelles distal to the ER, such as the golgi(e.g., cell surface immunoprecipitations or cell immunocytochemistryusing antibody specific for the protein, Examples I to III), forsecreted proteins, detecting the amount in the media, or detectingprotein transport through the various ER to golgi transport stages, forexample, by assaying polypeptides for the presence of complexoligosaccharide chains added by glycosyl transferases present in thegolgi. For example, as described in Example I and shown in FIGS. 1 and2, pulse-chase studies revealed that wild type CFTR maturation beginswithin an hour (acquires complex oligosaccharide chains resistant toendoglycosidase H digestion, i.e., “endo H resistant chains”), whereasexport-incompetent ΔF508 CFTR fails to mature even after two hours (doesnot acquire endoglycosidase H resistant chains; compare FIGS. 2A to 2C).Additional methods, both in vivo and in vitro, are known in the art thatcan be used for detecting an increase or decrease in intracellulartransport of a protein.

The isolated invention polypeptides having a substituted R of the R-X-Rwill generally be proteins that are naturally present in theintracellular transport apparatus during their maturation and aretherefore normally present on the cell surface, in lysosomes orendosomes, or are secreted by the cell. Thus, such polypeptides includecell surface ion channels, growth factors, immune response regulatorsreceptors (cytokines and growth factors including interleukins,interferons etc.), adhesion proteins (e.g., integrins and galectins),hormones, clotting factors, hemostatic effectors and receptors thereof.Nevertheless, other polypeptides that are not naturally present in theintracellular transport apparatus, but are modified so as to be presentin the intracellular transport apparatus, such as by attaching anappropriate targeting signal, also are included, so long as substitutedpolypeptide, as set forth herein, is exported to the cell surface in anamount greater than the unsubstituted polypeptide.

Polypeptides containing at least one arginine-framed tripeptide (AFT) inwhich at least one of the arginines has been substituted with anotheramino acid include export-incompetent polypeptides as well aspolypeptides for which no export deficiency is apparent (e.g., wild-typeproteins). Both naturally occurring and non-naturally occurringsubstituted polypeptides are included. Specific examples of polypeptidesthat can be substituted are ΔF508 CFTR polypeptides in which an arginineresidue at position 29, 516, 555, 766 has been substituted with lysine.Specific examples of polypeptides for which no export deficiency isapparent are wild-type CFTR's in which one or more arginines of thearginine-framed tripeptides have been substituted. For example,wild-type CFTR in which four arginines have been substituted with lysine(wild-type/4RK) appears to undergo more rapid maturation thanunsubstituted wild-type CFTR (compare FIGS. 2A to 2B). As withsubstituted export-incompetent CFTR, wild type CFTR polypeptides canhave one or more substitutions of the arginine at the indicatedpositions, or have multiply substituted arginines, in any combination.Additional export-incompetent CFTR polypeptides are known in the art andare specifically included when substituted as set forth herein.

The invention further includes polypeptides having minor variations,additions or deletions to the amino acid sequence of the polypeptidesdisclosed herein so long as the modified polypeptide has substantiallythe same biological activity or function as the unmodified polypeptide.As used herein, the term “substantially the same biological activity orfunction,” when used in reference to a modified polypeptide, means thatthe polypeptide retains sufficient biological activity associated withthe unmodified polypeptide as described herein or known in the art toprovide normal tissue function.

Modified polypeptides are therefore distinct from substitutedpolypeptides because modified polypeptides can have modificationsthroughout the polypeptide that do not destroy biological activity,whereas substituted polypeptides have one or more amino acids of theR-X-R sequence or a flanking sequence substituted with another aminoacid, where the substituted polypeptide is exported to the cell surfacein an amount or at a rate greater than the unsubstituted polypeptide.Thus, a substituted polypeptide as set forth herein can be modified solong as the polypeptide is exported from the ER in an amount or at arate greater than the unsubstituted polypeptide. For example, asubstituted export-incompetent CFTR polypeptide having modificationswould be exported from the ER in an amount or at a rate greater thanunsubstituted export-incompetent CFTR. Similarly, a substitutedwild-type CFTR having modifications would be exported to the cellsurface in an amount greater than unsubstituted wild-type CFTR.

Modified polypeptides that are “biologically active” or “functional” canbe identified through a functional assay. For example, modified CFTRwill exhibit substantial chloride efflux. An example of a functionalassay for a polypeptide having an AFT, would be the ability of themodified AFT to induce or increase transport of an export incompetentprotein. Additional functional assays for CFTR are known in the art andvarious assays for determining whether a modified polypeptide, such as acell surface or secreted protein, has a biological function or activityinclude ion transport, ligand binding, cell signaling, the ability tobind or interact with proteins in vitro or in vivo, enzymatic activity,gene activation or suppression and the ability to be modulated by agentsor proteins, for example.

As modified polypeptides will retain biological activity associated withunmodified polypeptide, modified polypeptides will generally have anamino acid sequence “substantially identical” to the amino acid sequenceof the unmodified polypeptide. As used herein, the term “substantiallyidentical,” when used in reference to a polypeptide, means that asequence of the polypeptide is at least 50% identical to a referencesequence. Modified polypeptides and substantially identical polypeptideswill have at least 70%, preferably 88%, more preferably 90%, and mostpreferably 95% homology to a reference polypeptide. For polypeptides,the length of comparison between sequences will generally be at least 15amino acids, preferably at least 25 amino acids, more preferably atleast 50 amino acids, and most preferably 100 amino acids or more.

As used herein, the terms “homology” or “homologous.” when used inreference to polypeptides, refers to the amino acid sequence similaritybetween two polypeptides. When an amino acid position in both of thepolypeptides is occupied by identical amino acids, then they arehomologous at that position. Thus, by “substantially homologous” ismeant an amino acid sequence that is largely, but not entirely,homologous, and which retains most or all of the activity as thesequence to which it is homologous.

Modified or substantially identical polypeptides can have one or moreadditions, deletions, or insertions, or non-conservative variations,located at positions of the amino acid sequence which do not destroy thefunction of the protein (as determined by functional assays, e.g., asdescribed herein) or conservative variations, for example, one aminoacid can be substituted for another of the same class (e.g., valine forglycine, arginine for lysine, etc.).

An example of an addition is a heterologous domain that imparts adistinct functionality upon the polypeptide. A heterologous domain canbe any small molecule, macromolecule or microfabricated device so longas it imparts an additional function. Particular heterologous domainsinclude those that provide a targeting function (e.g., an antibody,ligand, viral envelope protein), those that enhance or suppress activity(a derepressible or activatable moiety), and those that enablepurification (e.g., T7 tag, polyhistidine sequence etc.). The skilledartisan will know of other heterologous domains depending on theapplication and the distinct function desired.

An example of a deletion is where a small portion of the molecule isremoved. For example, deletion of an AFT may not alter CFTR biologicalactivity, but may induce or increase transport from the ER. Multipledeletions of AFTs may provide an additive or synergistic effect.Polypeptides having AFT deletions are specifically included so long asthe deleted polypeptide has increased transport in comparison toundeleted polypeptide.

As used herein, the term “conservative variation” denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the replacement ofa hydrophobic residue such as isoleucine, valine, leucine or methioninefor another, the replacement of a polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like.

Modified polypeptides further include “chemical derivatives,” in whichone or more of the amino acids therein has a side chain chemicallyaltered or derivatized. Such derivatized polypeptides include, forexample, amino acids in which free amino groups form aminehydrochlorides, p-toluene sulfonyl groups, carobenzoxy groups; the freecarboxy groups form salts, methyl and ethyl esters; free hydroxyl groupsthat form O-acyl or O-alkyl derivatives as well as naturally occurringamino acid derivatives, for example, 4-hydroxyproline for proline,5-hydroxylysine for lysine, homoserine for serine, ornithine for lysineetc. Also included are D-amino acids and amino acid derivatives that canalter covalent bonding, for example, the disulfide linkage that formsbetween two cysteine residues that produces a cyclized polypeptide.

The polypeptide modifications may be deliberate, as by site-directed(e.g., PCR based) or random mutagenesis (e.g., EMS) or may bespontaneous or naturally occurring. For example, naturally occurringallelic or splice variants can arise by alternative RNA splicing,polymorphisms or spontaneous mutations of a nucleic acid. Further,deletion of one or more amino acids can also result in a modification ofthe structure of the resultant polypeptide without significantlyaltering a biological activity. Deletion can lead to the development ofa smaller active molecule that may have broader utility. Those of skillin the art will recognize the numerous amino acids that can be modifiedor substituted with other chemically similar residues withoutsubstantially altering biological activity. All of the polypeptidesproduced by such modifications are included herein as long as themodified polypeptide possesses-most or all of a biological activity orfunction as described herein.

As the AFT's of CFTR are disclosed herein as contributing to ERretention or delay in maturation of proteins, the AFT sequences areuseful as competitive inhibitors of ER retention. Thus, in anotherembodiment, the present invention provides isolated polypeptides havingan AFT sequence. The amino acid length of polypeptides that contain theAFT can range from about 4 amino acids up to about 50 amino acids.Preferably, the amino acid length is from about 5 to about 40 aminoacids, more preferably, from about 6 to about 30 amino acids and, mostpreferably, from about 7 to about 25 amino acids. For example, thepolypeptide can have the sequence X₁-R-X₂-R-X₃ where X₁, X₂ and X₃ areany amino acid. In certain aspects, the X₂ amino acid is not alanine,asparagine or glutamate.

Exemplary polypeptides having an AFT sequence are heptamers based on theAFT sequence region of the corresponding CFTR polypeptide. Inparticular, GYRQRLE (SEQ ID NO:1), which is based on the sequenceSWTRPILRKGYRQRLELSDIYQIPS (SEQ ID NO:6); EYRYRSV (SEQ ID NO:2), which isbased on the sequence NIIFGVSYDEYRYRSVIKACQLEED (SEQ ID NO:7); GQRARIS(SEQ ID NO:3, which is based on the sequence GEGGITLSGGQRARISLARAVYKDA(SEQ ID NO:8); and QARRRQS (SEQ ID NO:4), which is based on the sequenceSVISTGPTLQARRRQSVLNLMTHSV (SEQ ID NO:9). It is understood that thestandard single letter amino acid abbreviation is used to denote theamino acids of the invention polypeptides (see for example, Zubay, G.L., Biochemistry page 12, Addison-Wesely Publishing, Inc., 1983).

AFT containing polypeptides can have multiple AFT sequences, if desired.AFT polypeptides of the invention also can be modified as set forthherein so long as a biological activity or function is substantially thesame as unmodified polypeptide. Additional modifications furtherinclude, for example, those that enhance or increase activity, so as tobe more effective inhibitors of ER retention.

The polypeptides of the invention can be prepared by a variety ofmethods known in the art, such as by purification from an appropriateorganism or cell using typical biochemical methods (e.g., columnchromatography), by chemical synthesis (peptide synthesizers, e.g.Applied Biosystems, Inc., Model 540A (Foster City, Calif.)), byexpression screening (using an antibody that binds to the polypeptide).An example of one means is by expression of a nucleic acid in a cellencoding an invention polypeptide in a host cell, such as bacteria,yeast or mammalian cell, and purifying the expressed polypeptide usingmethods known in the art. Other well known methods are described inDeutscher et al., (Guide to Protein Purification: Methods in EnzymologyVol. 182, Academic Press (1990)).

The invention further provides isolated nucleic acids encoding inventionpolypeptides, fragments thereof, complementary sequences thereto, andantisense sequences thereof. In one embodiment, invention isolatednucleic acids encode export incompetent CFTR's in which an R of theR-X-R motif is substituted, for example, with lysine. In anotherembodiment, invention isolated nucleic acids encode wild-type CFTR's inwhich an R of the R-X-R motif is substituted, for example, with lysine.In certain other aspects, nucleic acids encoding polypeptides having anAFT sequence also are provided.

As used herein, the terms “nucleic acid,” “polynucleotide,”“oligonucleotide,” or “primer” are used interchangeably to refer todeoxyribonucleic acid (DNA) or ribonucleic (RNA), either double orsingle stranded, linear or circular. RNA can be unspliced or splicedmRNA, rRNA, tRNA or antisense RNA. DNA can be complementary DNA (cDNA),genomic DNA, or an antisense. Specially included are nucleotideanalogues and derivatives, such as those used to provide resistance todegradation by nucleases, which can function as antisense or encodeinvention polypeptides.

An “isolated” or “substantially pure” nucleic acid means that thenucleic acid is not immediately contiguous with the coding sequenceswith either the 5′ end or the 3′ end with which it is immediatelycontiguous in the naturally occurring genome of the organism from whichit is derived. The term therefore includes, for example, a recombinantDNA that is a separate molecule independent of other sequences (e.g., acDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) as well as a recombinant DNA incorporated into avector, an autonomously replicating plasmid or virus; or a genomic DNAof a prokaryote or eukaryote. It also includes a recombinant DNA that ispart of a hybrid or fusion, for example, a gene encoding an additionalpolypeptide sequence. The term therefore does not include nucleic acidspresent among millions of sequences in a genomic or cDNA library, or ina restriction digest of a library fractionated on a gel.

The nucleic acids of the invention also include nucleic acids that aredegenerate as a result of the genetic code. There are 20 natural aminoacids, most of which are specified by more than one codon. Degeneratesequences may not selectively hybridize to other invention nucleicacids; however, they are nonetheless included as they encode inventionpolypeptides, arginine-framed tripeptide repeats and fragments thereof.

The invention also includes nucleic acids substantially homologous withthe nucleic acids encoding invention polypeptides. As used herein, theterm “homologous,” when used in reference to nucleic acid molecule,refers to similarity between two nucleotide sequences. When a nucleotideposition in both of the DNA molecules is occupied by identicalnucleotides, then they are homologous at that position. Preferably,“substantially homologous” nucleic acid sequences are at least 70%homologous, more preferably at least 80% homologous and most preferably90% homologous, and retains the biological activity associated with thesequence to which it is homologous. For nucleic acids, the length ofcomparison between sequences will generally be at least 30 nucleotides,preferably at least 50 nucleotides, more preferably at least 75nucleotides, and most preferably 110 nucleotides or more. Algorithms foridentifying homologous sequences that account for sequence gaps,mismatches, their length and location, are known in the art, such asBLAST (see e.g., Altschul et al., J. Mol. Biol. 215:403–10, 1990).

The invention nucleic acids are useful for encoding inventionpolypeptides, when such nucleic acids are incorporated into expressionsystems disclosed herein or known in the art. In addition, inventionnucleic acids are useful as probes which can be used to identify thepresence of a nucleic acid related to an invention nucleic acid or todetect the presence or an amount of DNA or mRNA in a sample, for example(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y., 1989). Such probes can be modified so as to bedetectable using radionuclides, luminescent moieties etc.

The invention further includes nucleic acid sequences complementary tothe sequences described herein, such as antisense. Such single or doublestranded RNA sequences (known as “RNA”) are useful for inhibiting geneexpression, for example (Kennerdell et al. (1998) Cell 95:1017–1026;Fire et al. (1998) Nature 391:806–811). Antisense sequences caninterfere with a nucleic acid encoding a factor that mediates orcontributes to ER retention (e.g., chaperones such as calnexin, hsc70etc.). An effective amount of an antisense from the coding region of afactor that mediates or contributes to ER retention can be useful fortreating physiological disorders associated with export-incompetentproteins including CFTR, as described herein.

Invention nucleic acid sequences can be obtained using various standardtechniques known in the art (e.g., molecular cloning, chemicalsynthesis) and the purity can be determined by polyacrylamide or agarosegel electrophoresis; DNA sequencing and the like. Nucleic acids also canbe isolated using hybridization or computer-based techniques, which arewell known in the art. Such techniques include, but are not limitedto: 1) hybridization of genomic DNA or cDNA libraries with probes todetect homologous nucleotide sequences; 2) antibody screening ofpolypeptides expressed by DNA sequences (e.g., using an expressionlibrary); 3) polymerase chain reaction (PCR) of genomic DNA or cDNAusing primers capable of annealing to a nucleic acid sequence ofinterest; 4) computer searches of sequence databases for relatedsequences; and 5) differential screening of a subtracted nucleic acidlibrary.

The nucleic acids of the invention can, if desired, be naked or be in acarrier suitable for passing through a cell membrane (e.g., DNA-liposomecomplex), contained in a vector (e.g., retroviral vector, adenoviralvectors and the like), or linked to inert beads or other heterologousdomains (e.g., antibodies, biotin, streptavidin, lectins, etc.), orother appropriate compositions disclosed herein. Thus, both viral andnon-viral means of nucleic acid delivery can be achieved and arecontemplated. The nucleic acids of the invention also can containadditional nucleic acid sequences linked thereto that encodes apolypeptide having a distinct functionality, such as the heterologousdomains set forth herein. Invention polynucleotides also can bemodified, for example, to be resistant to nucleases to enhance theirstability in a pharmaceutical formulation, for example.

For propagation or expression in cells, invention nucleic acids can beinserted into a vector. The term “vector” refers to a plasmid, virus orother vehicle known in the art that can be manipulated by insertion orincorporation of a nucleic acid. Such vectors can be used for geneticmanipulation (i.e., “cloning vectors”) or can be used to transcribe ortranslate the inserted polynucleotide (i.e., “expression vectors”). Avector generally contains at least an origin of replication forpropagation in a cell and a promoter. Control elements, includingpromoters, present within an expression vector are included tofacilitate proper transcription and translation (e.g., splicing signalfor introns, maintenance of the correct reading frame of the gene topermit in-frame translation of mRNA and, stop codons etc.).

In vivo or in vitro expression of the invention nucleic acids can beconferred by a promoter operably linked to the nucleic acid. “Promoter”refers to a minimal nucleic acid sequence sufficient to directtranscription of the nucleic acid to which the promoter is operablylinked (see e.g., Bitter et al., Methods in Enzymology 153:516–544,1987). Promoters can constitutively direct transcription, can betissue-specific, or can render inducible or repressible transcription;such elements are generally located in the 5′ or 3′ regions of thenative gene.

A “tissue-specific promoter” means a promoter that is active inparticular cells or tissues which therefore confers transcription of theoperably linked nucleic acid in the particular cells, e.g., liver cells,hematopoietic cells, or cells of a specific tissue within an animal,e.g., pancreatic βcells. The term also covers so-called “leaky”promoters, which regulate expression of a selected DNA primarily in onetissue, but cause expression in other tissues as well. An “induciblepromotor” means a promoter whose activity level increases in response totreatment with an external signal or agent (e.g., metallothionein IIApromoter, heat shock promoter). A “repressible promotor” or “conditionalpromoter” means a promoter whose activity level decreases in response toa repressor or an equivalent compound. When the repressor is no longerpresent, transcription is activated or derepressed. Such promoters maybe used in combination and also may include additional DNA sequencesthat are necessary for transcription and expression, such as introns andenhancer sequences.

As used herein, the term “operably linked” means that a selected nucleicacid (e.g., encoding a substituted export-incompetent CFTR) and aregulatory sequence(s) are connected in such a way as to permittranscription when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s). Typically,a promoter is in close proximity and 5′ of the nucleic acid to allow thepromoter to regulate expression of the nucleic acid. However, indirectoperable linkage is also possible when a promoter on a first plasmidcontrols expression of a protein that, in turn, regulates a promotercontrolling expression of the nucleic acid on a second plasmid.

When cloning in bacterial systems, constitutive promoters such as T7 andthe like, as well as inducible promoters such as pL of bacteriophage λplac, ptrp, ptac ptrp-lac hybrid promoter) may be used. When cloning inmammalian cell systems, constitutive promoters such as SV40, RSV and thelike, or inducible promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., themouse mammary tumor virus long terminal repeat, the adenovirus latepromoter) may be used. Promoters produced by recombinant DNA orsynthetic techniques may also be used to provide for transcription ofthe nucleic acid sequences of the invention.

Mammalian expression systems which utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, the nucleic acid sequence may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. Alternatively, thevaccinia virus 7.5K promoter may be used. (see e.g., Mackett et al.(1982) Proc. Natl. Acad. Sci. USA 79:7415–7419; Mackett et al. (1984) J.Virol. 49:857–864; and Panicali et al. (1982) Proc. Natl. Acad. Sci. USA79:4927–4931).

Mammalian expression systems further include vectors specificallydesigned for “gene therapy” methods including adenoviral vectors (U.S.Pat. Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat.No. 5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979)and retroviral vectors (U.S. Pat. Nos. 5,624,820, 5,693,508 and5,674,703 and WIPO publications WO 92/05266 and WO92/14829). Vectorsbased on bovine papilloma virus (BPV) have the ability to replicate asextrachromosomal elements (Sarver et al., (1981) Mol. Cell. Biol.1:486). Shortly after entry of an extrachromosomal vector into mousecells, the vector replicates to about 100 to 200 copies per cell.Because transcription of the inserted cDNA does not require integrationof the plasmid into the host's chromosome, a high level of expressionoccurs. Such vectors also have been employed in gene therapy (U.S. Pat.No. 5,719,054). CMV based vectors also are included (U.S. Pat. No.5,561,063).

In yeast, a number of vectors containing constitutive or induciblepromoters may be used (see e.g., Current Protocols in Molecular Biology,Vol. 2, Ch. 13, Ed. Ausubel et al., Greene Publish. Assoc. & WileyInterscience, 1988; Grant et al., “Expression and Secretion Vectors forYeast,” in Methods in Enzymology, Vol. 153, pp. 516–544, Eds. Wu &Grossman, 31987, Acad. Press, N.Y., 1987; Glover, DNA Cloning Vol. II,Ch. 3, IRL Press, Wash., D.C., 1986; Bitter, “Heterologous GeneExpression in Yeast,” Methods in Enzymology Vol. 152, pp. 673–684, Eds.Berger & Kimmel, Acad. Press, N.Y., 1987; and The Molecular Biology ofthe Yeast Saccharomyces, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II 1982). A constitutive yeast promoter such as ADHor LEU2 or an inducible promoter such as GAL may be used (“Cloning inYeast,” R. Rothstein In: DNA Cloning, A Practical Approach, Vol. 11, Ch.3, Ed. D. M. Glover, IRL Press, Wash., D.C., 1986). Alternatively,vectors that facilitate integration of foreign nucleic acid sequencesinto a yeast chromosome, via homologous recombination for example, areknown in the art and can be used. Yeast artificial chromosomes (YAC) aretypically used when the inserted polynucleotides are too large for moreconventional yeast expression vectors (e.g., greater than about 12 kb).

Thus, in accordance with the present invention, nucleic acids encodinginvention polypeptides may be inserted into an expression vector forexpression in vitro (e.g., using in vitro transcription/translationkits, which are available commercially), or may be inserted into anexpression vector that contains a promoter sequence which facilitatestranscription in either prokaryotes or eukaryotes by transfer of anappropriate nucleic acid into a suitable cell, organ, tissue ororganism.

As used herein, a “transgene” is any piece of nucleic acid that isinserted by artifice into a host cell, and becomes part of the organismthat develops from that cell. A transgene can include one or morepromoters and any other DNA, such as introns, necessary for expressionof the selected DNA, all operably linked to the selected DNA, and mayinclude an enhancer sequence. A transgene may include a gene that ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Transgenes may integrate into the host cells genome or bemaintained as a self-replicating plasmid (e.g., yeast 2μ plasmids).

As used herein, a “host cell” is a cell into which a nucleic acid isintroduced that can be propagated, transcribed, or encoded polypeptideexpressed. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication.

Host cells include but are not limited to microorganisms such asbacteria, yeast, insect and mammalian cells. For example, bacteriatransformed with recombinant bacteriophage nucleic acid, plasmid nucleicacid or cosmid nucleic acid expression vectors; yeast transformed withrecombinant yeast expression vectors; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid); insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus); or animal cellsystems infected with recombinant virus expression vectors (e.g.,retroviruses, adenovirus, vaccinia virus), or transformed animal cellsystems engineered for stable expression.

For long-term expression of invention polypeptides, stable expression ispreferred. Thus, using expression vectors that contain viral origins ofreplication, for example, cells can be transformed with a nucleic acidcontrolled by appropriate control elements (e.g., promoter/enhancersequences, transcription terminators, polyadenylation sites, etc.).Although not wishing to be bound or so limited by any particular theory,stable maintenance of expression vectors in mammalian cells is believedto occur by integration of the vector into a chromosome of the hostcell. Optionally, the expression vector also can contain a nucleic acidencoding a selectable or identifiable marker conferring resistance to aselective pressure thereby allowing cells having the vector to beidentified, grown and expanded. Alternatively, the selectable marker canbe on a second vector that is cotransfected into a host cell with afirst vector containing an invention polynucleotide.

A number of selection systems may be used, including, but not limited tothe herpes simplex virus thymidine kinase gene (Wigler et al. (1977)Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase gene(Szybalska et al., Proc. Natl. Acad. Sci. USA 48:2026, 1962), and theadenine phosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genescan be employed in tk-, hgprt⁻ or aprt⁻ cells respectively.Additionally, antimetabolite resistance can be used as the basis ofselection for dhfr, which confers resistance to methotrexate (Wigler etal. (1980) Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al. (1981)Proc. Natl. Acad. Sci. USA 78:1527); the gpt gene, which confersresistance to mycophenolic acid (Mulligan et al. (1981) Proc. Natl.Acad. Sci. USA 78:2072; the neomycin gene, which confers resistance tothe aminoglycoside G-418 (Colberre-Garapin et al. (1981) J. Mol. Biol.150:1); and the hygromycin gene, which confers resistance to hygromycin(Santerre et al. (1984) Gene 30:147). Recently, additional selectablegenes have been described, namely trpB, which allows cells to utilizeindole in place of tryptophan; hisD, which allows cells to utilizehistinol in place of histidine (Hartman et al. (1988) Proc. Natl. Acad.Sci. USA 85:8047); and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory, ed.,1987).

As used herein, the term “transformation” means a genetic change in acell following incorporation of DNA (e.g., a transgene) exogenous to thecell. Thus, a “transformed cell” is a cell into which, or a progeny ofwhich, a DNA molecule has been introduced by means of recombinant DNAtechniques.

Transformation of a host cell with DNA may be carried out byconventional techniques known to those skilled in the art. For example,when the host cell is a eukaryote, methods of DNA transformationinclude, for example, calcium phosphate co-precipitates, conventionalmechanical procedures such as microinjection, electroporation, insertionof a plasmid encased in liposomes, and viral vectors. Eukaryotic cellsalso can be cotransformed with invention nucleic acid sequences orfragments thereof, and a second DNA molecule encoding a selectablephenotype, as those described herein. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein (see e.g., Eukaryotic Viral Vectors, Cold SpringHarbor Laboratory, Gluzman ed., 1982). When the host is prokaryotic(e.g., E. coli), competent cells which are capable of DNA uptake can beprepared from cells harvested after exponential growth phase andsubsequently treated by the CaCl₂ method using procedures well known inthe art. Transformation of prokaryotes also can be performed byprotoplast fusion of the host cell.

In another embodiment, the invention provides non-human transgenicanimals having invention nucleic acids. As used herein, the term“animal,” when modified by the term “transgenic,” refers to an organismthat reproduces sexually. Transgenic animals can be produced by methodsknown in the art.

The term “transgenic animal” refers to any animal whose somatic or germline cells bear genetic information received, directly or indirectly, bydeliberate genetic manipulation at the subcellular level, such as bymicroinjection or infection with recombinant virus. The term “trangenic”further includes cells or tissues (i.e., “transgenic cell,”“transgenictissue”) obtained from a transgenic animal genetically manipulated asdescribed herein. In the present context, a “transgenic animal” does notencompass animals produced by classical crossbreeding or in vitrofertilization, but rather denotes animals in which one or more cellsreceive a recombinant DNA molecule. Invention transgenic animals can beeither heterozygous or homozygous with respect to the transgene. Methodsfor producing transgenic animals are well known in the art (see forexample, U.S. Pat. Nos. 5,721,367; 5,695,977; 5,650,298 and 5,614,396).

Preferred transgenic animals contain the transgene integrated into germcells. Transgenic animals having a transgene integrated into germ cellshave the ability to transfer the transgene to offspring. If suchoffspring in fact possess some or all of the transgene, then they, too,are transgenic animals. Homologous recombination is one mechanism inwhich a transgene is stably inserted into the genome. Although it isfurther preferred that the transgene be integrated into the animal'schromosome, the present invention also contemplates the use ofextrachromosomally replicating sequences containing a transgene, such asthose similar to yeast artificial chromosomes.

In the transgenic animals described herein, the transgene encodes asubstituted polypeptide, such as CFTR, for example, or a peptide havingan AFT sequence. A particularly useful transgenic would be an animalthat exhibits characteristics of cystic fibrosis (e.g., that expressesan export-incompetent CFTR) which has been transformed with a transgeneencoding a substituted CFTR or a polypeptide having an AFT sequence.Expression of the transgene can cause animal cells, such as airpassageway epithelial cells or pancreatic cells, to increase CFTRexpression on the cell surface. Animals that express otherexport-incompetent polypeptides can be transformed with a polypeptidehaving an AFT sequence, which can induce or increase intracellulartransport of the export-incompetent cell surface polypeptide or secretedpolypeptide. Alternatively, transgenic animals transformed with anantisense nucleic acid capable of inhibiting translation of an ERretention factor can exhibit increased export of export-incompetent orother proteins that utilize the intracellular transport pathway. Anyanimal that can be produced by transgenic technology is included in theinvention, although mammals are preferred which include non-humanprimates, sheep, goats, horses, cattle, pigs, rabbits, and rodents suchas mice, guinea pigs, hamsters, rats and gerbils.

Invention polypeptides can be used to generate additional reagents, suchas antibodies. Thus, in accordance with the present invention,antibodies that bind to CFTR polypeptides having substitutions as setforth herein, fragments thereof and polypeptides having an AFT sequenceare provided. Antibody comprising polyclonal antibodies, pooledmonoclonal antibodies with different epitopic specificities, anddistinct monoclonal antibody preparations, are provided. The inventionantibodies can be used in diagnostic methods, purification methods andin the treatment methods, as disclosed herein.

The term “antibody” includes intact molecules as well as fragmentsthereof, such as Fab, F(ab′)2, and Fv which are capable of binding to anepitopic determinant present in an invention polypeptide. Other antibodyfragments are included so long as the fragment retains the ability toselectively bind with its antigen.

As used herein, the term “epitope” refers to an antigenic determinant onan antigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Generally, epitopes have at least fivecontiguous amino acids.

Antibodies that bind to invention polypeptides can be prepared usingintact polypeptide or small peptide fragments thereof as the immunizingantigen. The polypeptide used to immunize an animal is derived fromtranslated DNA or chemically synthesized, if desired, can be conjugatedto a carrier protein. Such commonly used carriers that are chemicallycoupled to the immunizing peptide include, for example, keyhole limpethemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanustoxoid.

Monoclonal antibodies are made by methods well known to those skilled inthe art (Kohler et al., Nature 256:495, 1975; and Harlow et al.,Antibodies: A Laboratory Manual, page 726, Cold Spring Harbor Pub.,1988). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, verifying the presence ofantibody production by analyzing a serum sample, removing the spleen toobtain B lymphocytes, fusing the B lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures. Monoclonal antibodies can be isolated andpurified from hybridoma cultures by a variety of well-establishedtechniques which include, for example, affinity chromatography withProtein-A Sepharose, size-exclusion chromatography, and ion-exchangechromatography (see e.g., Coligan et al., “Production of PolyclonalAntisera in Rabbits, Rats, Mice and Hamsters,” in: Current Protocols inImmunology sections 2.7.1–2.7.12 and sections 2.9.1–2.9.3; and Barnes etal., “Purification of Immunology G (IgG),” in: Methods in MolecularBiology, Vol. 10, pages 79–104, Humana Press, 1992).

The preparation of polyclonal antibodies is well-known to those skilledin the art (see, e.g., Green et al., “Production of PolyclonalAntisera,” in: Immunochemical Protocols, pages 1–5, Manson, ed., HumanaPress, 1992; Harlow et al., 1988, supra; and Coligan et al., 1992,supra, section 2.4.1).

Antibody fragments (e.g., Fab, F(ab′)₂, and Fv) of the present inventioncan be prepared by proteolytic hydrolysis of the antibody, for example,by pepsin or papain digestion of whole antibodies.

In accordance with the present invention, there are provided methods foridentifying a therapeutic agent for treating cystic fibrosis. A methodof the invention comprises contacting a cell expressing anexport-incompetent CFTR with a test agent under conditions allowing aninteraction between the agent and a factor mediating or contributing toexport-incompetence, and determining whether the agent increases theamount of export-incompetent CFTR on the cell surface, where anincreased amount of CFTR on the cell surface identifies a therapeuticagent for treating cystic fibrosis. Particular export-incompetent CFTRsthat may be used include ΔF508 and R1066C.

In another embodiment, the invention provides methods for identifying anagent that induces or increases transport of an export-incompetentprotein. A method of the invention comprises contacting anexport-incompetent protein with a test agent under conditions allowingan interaction between the agent and a factor mediating or contributingto export-incompetence, and determining whether the agent increases theamount or the rate of protein transported, where an increased amount ofthe protein transported identifies an agent that induces or increasestransport of an export-incompetent protein. In one aspect, the method isperformed using a cell expressing an export-incompetent protein. Inanother aspect, the method is performed in vitro. In certain additionalaspects, the export-incompetent protein is a cell surface protein, asecreted protein, a lysosomal protein or an endosomal protein. Such invitro assays are described, for example, in Zhang et al. (Nat Struct.Biol. 5:180–183 (1998))

As used herein, the terms “transport” or “intracellular transport”describes the movement or progression of a molecule along the ER-golgipathway. Thus, an induction or increase in “transport” means that theprotein is transported from the ER to the cis-golgi, or beyond, e.g., tothe medial-golgi, to the trans-golgi, to the cell surface or, asappropriate, secreted or, to the endosome or lysosome. It isspecifically intended that the term transport involve movement from anypoint within the pathway to any point within the pathway. Preferably, anagent that induces or increases transport will promote movement of theprotein to its final destination. For example, an agent that induces orincreases CFTR transport will promote movement from the ER to the cellsurface. Thus, an “increased rate of transport” or an “increase in therate of transport” means that the time needed for transport is decreasedand an “increase in an amount transported” means that the amount ofprotein transported is greater.

In the methods of the invention for identifying a therapeutic agent fortreating CFTR, or for identifying an agent that induces or increasestransport of an export-incompetent protein, an increase in an amount ofthe protein on the cell surface or an increase in an amount of proteintransported will be detected. Various detection methods can be employed.For example, to detect an increase in an amount of protein on the cellsurface, immunostaining with a specific antibody (e.g., anti-CFTR,M3A7), or direct visualization (e.g., a CFTR-GFP fusion) as disclosedherein, can be employed. Additional methods useful for determiningwhether there is an increase in cell surface protein included cellpanning. In cell panning assays, plates are coated with an antibody thatbinds to the cell surface protein. The number of cells that binds to theantibody coated plate corresponds to an amount of protein on the cellsurface.

To detect an increase in an amount of protein transported, methods thatdetect an increased amount of protein on the cell surface can beemployed. Additionally, as disclosed herein, protein transport can bedetected by the acquisition or removal of particular oligosaccharidechains. For example, endoH resistant oligosaccharide chains are acquiredin the golgi. Thus, detection of endoH resistant chains provides a meanswith which to detect transport of protein from the ER to golgi. Laterstages of transport (e.g., trans-golgi) are associated with the removalor addition of particular oligosaccharides from proteins. Thus, otherglycosidases, such as endoglycosidase F, neuraminindase and the like canbe used to detect whether an increase in protein transport to laterstage, s such as the trans-golgi, for example, occurs. Such enzymes arecommercially available (e.g., Boehringer Manheim Biochemicals.Indianapolis, Ind.) or are otherwise known in the art.

Polypeptide sequence motifs that mediate or contribute to ER retentionof export-incompetent CFTR are disclosed herein. When cells that expressexport-incompetent ΔF508 CFTR are treated with polypeptides having asequence motif such as R-X-R, an increase in CFTR transport from the ERto the cell surface is observed (see for example, FIG. 4).

Thus, in accordance with the present invention, there are providedmethods for treating cystic fibrosis. A method of the inventioncomprises administering to a subject a pharmaceutical formulationcomprising a polypeptide having an arginine framed peptide sequence inan amount effective for treating cystic fibrosis. In one aspect, thesubject expresses an export-incompetent CFTR, such as ΔF508 or R1066CCFTR. In another embodiment, a method of the invention comprisesadministering to a subject a pharmaceutical formulation containing aCFTR, wherein at least one arginine of the arginine framed peptidesequence is substituted with an amino acid, in an amount effective fortreating cystic fibrosis. The substituted CFTR can be wild-type CFTR,mutated CFTR, can be further modified, and can be encoded by a nucleicacid sequence, as disclosed herein. For example, a nucleic acid encodinga CFTR polypeptide having one or more amino acid substitutions of anR-X-R sequence can be administered to a subject afflicted with CF inorder to treat CF. Other CFTR mutant sequences are known including, forexample, ΔI507, N1303K, S549I, S549R, A559T, H139R, G149R, D192G, R258G,S949L, H949Y, H1054D, G1061R, L1065P, R1066C, R1066H, R1066L, Q1071P, L1077P, H1085R, W1098R, M1101K, M1101R, and can similarly be modified orsubstituted as set forth herein.

The fact that other proteins destined for the intracellular transportpathway frequently exhibit export incompetency due to mutations, orother factors, indicates that the transport of such export-incompetentpolypeptides can be induced or increased by treating with a polypeptidehaving an AFT sequence. Accordingly, physiological disorders associatedwith export-incompetent proteins can similarly be treated.

Thus, in accordance with the present invention, there are providedmethods for treating a subject having a physiological disorderassociated with an export incompetent protein. A method of the inventioncomprises administering to the subject a pharmaceutical formulationcomprising a polypeptide having an R-X-R sequence in an amount effectivefor treating a physiological disorder associated with anexport-incompetent protein. In one aspect, the polypeptide has at leastfour amino acids or, in another aspect, at least seven amino acids. Incertain other aspects of the invention method, the export incompetentprotein is a cell surface protein, a secreted protein, a lysosomalprotein or an endosomal protein.

Physiological disorders associated with an export incompetent proteinthat can be treated in a method of the invention include, for example,Stargardt's disease and particular types of macular dystrophy caused bymutations of the retinal rod transporter, ABC-R, resulting in deficiencyof ER export. Additional candidate physiological disorders associatedwith various other ABC (adenine-nucleotide binding cassette) proteins.

The methods for treating a subject having a physiological disorderassociated with an export incompetent protein can be practiced with theinvention polypeptides having an AFT sequence based on the CFTRsequence, such as GYRQRLE (SEQ ID NO:1), EYRYRSV (SEQ ID NO:2), GQRARIS(SEQ ID NO:3) and QARRRQS (SEQ ID NO:4). Alternatively, the methods canbe practiced using polypeptides having AFT sequence, including flankingsequences, if desired, based upon the amino acid sequence of the exportincompetent polypeptide that causes or is associated with thephysiological disorder being treated. In this way, increased specificitycan be provided by using polypeptides having an AFT polypeptidessequence based on the polypeptide sequence. Increased specificity maydecrease potentially deleterious side effects caused by inducing orincreasing ER export of unrelated polypeptides, for example, byinhibiting the interaction of an ER retention factor with the unrelatedprotein thereby increasing its export.

As the invention polypeptides and nucleic acids are useful for treatingphysiological disorders associated with an export-incompetent protein,including cystic fibrosis, the present invention also providespharmaceutical formulations comprising invention polypeptides andnucleic acids.

The compositions administered to a subject will be in a“pharmaceutically acceptable” or “physiologically acceptable”formulation. As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, diluents, excipients andthe like that can be administered to a subject, preferably withoutexcessive adverse side effects (e.g., nausea, headaches, etc.). Suchpreparations for administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Vehicles includesodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's, or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobial,anti-oxidants, chelating agents, and inert gases and the like. Variouspharmaceutical formulations appropriate for administration to a subjectknown in the art are applicable in the methods of the invention (e.g.,Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.,Easton, Pa., 1990 and The Merck Index, 12th ed., Merck Publishing Group,Whitehouse, N.J., 1996).

Controlling the duration of action or controlled delivery of anadministered composition can be achieved by incorporating thecomposition into particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate,methylcellulose, carboxymethylcellulose, protamine sulfate, orlactide/glycolide copolymers, polylactide/glycolide copolymers, orethylenevinylacetate copolymers. The rate of release of the compositionmay be controlled by altering the concentration or composition of suchmacromolecules. For example, it is possible to entrap an AFT sequence inmicro-capsules prepared by coacervation techniques or by interfacialpolymerization, for example, by the use of hydroxymethylcellulose orgelatin-microcapsules or poly(methylmethacrolate) microcapsules,respectively, or in a colloid drug delivery system. Colloidal dispersionsystems include macromolecule complexes, nano-capsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes.

The compositions administered by a method of the invention can beadministered parenterally by injection or by gradual perfusion overtime. The composition can be administered via inhalation, intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity, ortransdermally, and preferably is administered intravascularly. Thecompositions can be administered in a single dose, or multiple doses.The doses needed for treating a physiological disorder associated withan export-incompetent protein will be sufficient to ameliorate some orall of the symptoms of the disorder. Such dosages can readily bedetermined by those skilled in the art (see for example, Ansel et al.Pharmaceutical Drug Delivery Systems, 5th ed. (Lea and Febiger (1990),Gennaro ed.).

The AFT polypeptides increase export incompetent CFTR cell surfaceexpression. Although not wishing to be bound by any particular theory,it is reasonable to assume that the mechanism by which the AFTpolypeptide sequence increases export is by competitively inhibitingbinding of an ER retention factor to its recognition site. As usedherein, the term “ER retention factor” refers to any biologicalmolecule, such as a protein, macromolecule, macromolecular machinery ororganelle that mediates or contributes to a protein's retention in theER, as assessed by an inability or delay in active or passive export oregress of the protein from the ER.

It is possible that such an ER retention factor may also recognizesignals other than an AFT. Thus, even though an export-incompetentprotein lacks an AFT sequence, polypeptides having an AFT sequence maystill competitively inhibit an ER retention factor thereby inducing orincreasing transport of a protein that does not have an AFT sequence.

Thus, in another embodiment, the invention provides methods for inducingor increasing intracellular transport of an export-incompetent proteinin a cell. A method of the invention comprises contacting a cell with acomposition comprising a polypeptide having an R-X-R sequence in anamount sufficient for inducing or enhancing intracellular transport ofthe export-incompetent protein. In one aspect, the export-incompetentprotein has an AFT sequence. In certain other aspects, the exportincompetent protein is secreted or is a cell surface protein.

Any cell can be employed in a method of the invention for identifying aCF therapeutic agent or for identifying an agent that induces orincreases transport of an export-incompetent protein, so long as thecell expresses or is made to express an export-incompetent protein.

In another embodiment, there are provided methods for identifying anagent that inhibits or disrupts an interaction between an R-X-Rpolypeptide and an ER retention factor. A method of the inventioncomprises incubating an R-X-R polypeptide and an ER retention factorunder conditions allowing their interaction; adding a test agent to theincubation; and detecting binding between said R-X-R polypeptide andsaid ER retention factor, where decreased binding in the presence of thetest agent identifies an agent that inhibits or disrupts an interactionbetween an R—X-R polypeptide and an ER retention factor.

As used herein, the term “incubating” refers to conditions that allowthe contact, binding or interaction between a polypeptide, functionalfragment or polynucleotides encoding same and the test agent. The term“contacting” as used herein includes in solution, in solid phase, incells and in an animal.

A therapeutic agent for treating CFTR or other physiological disordersassociated with an export-incompetent protein, as well as agents thatinduce or increase transport of an export-incompetent protein and agentsthat inhibit, prevent or disrupt the interaction between an R-X-Rpolypeptide and an ER retention factor can essentially be any molecule.Generally the agent will be a small organic molecule (e.g., a peptide)capable of interacting with either an appropriate polypeptide or ERretention factor such that the agent competitively or non-competitivelyinhibits, interferes with or antagonizes, partially or completely, theinteraction. An example of a competitive antagonist would be an agentthat binds to an ER retention factor at the site of interaction with thepolypeptide that subsequently prevents interaction with the polypeptide.Alternatively, a competitive antagonist can bind to an ER retentionsignal present on a polypeptide, such as an AFT, with which the ERretention factor interacts, thereby preventing or inhibiting itsinteraction with an ER retention factor. An example of non-competitiveantagonist would be an agent that binds either an ER retention factor ora polypeptide containing an appropriate retention signal, but does notprevent or inhibit its interaction. Nevertheless, such a non-competitiveantagonist prevents or inhibits ER retention of the complex, therebypromoting export.

Although such agents will generally directly interact with the ERretention factor or the polypeptide with which the ER retention factorinteracts, agents that function indirectly also are contemplated. Forexample, an indirect agent may activate or increase an interactionbetween a polypeptide and a factor that contributes to or mediates ERexport, thereby inducing or increasing transport from the ER

Further included are agents that function through any enzymaticinteraction in which the agent directly or indirectly performs abiochemical modification (e.g., phosphorylation) of the polypeptide orER retention factor that results in inhibiting or blocking ER retention.For example, post-translational modification of an amino acid sequencenear an AFT sequence may prevent or inhibit its interaction with an ERretention factor. Thus, agents that directly or indirectly alterpolypeptides, such as to increase or decrease phosphorylation,ubquitination, glycosylation, proteolytic cleavage and the like aretherefore included.

AFT polypeptides also can be employed to identify an ER retentionfactor. Thus, the invention further provides a method for identifying anER retention factor. A method of the invention comprises contacting acomposition suspected of containing an ER retention factor with apolypeptide having an R-X-R sequence under conditions allowing theirinteraction and detecting binding between the factor and thepolypeptide, thereby identifying an ER retention factor. The method canbe performed in vitro. The method also can employ polypeptides havingvarious lengths as set forth herein. Specific polypeptides includeGYRQRLE (SEQ ID NO:1), EYRYRSV (SEQ ID NO:2), GQRARIS (SEQ ID NO:3) andQARRRQS (SEQ ID NO:4).

In various other embodiments, the invention also provides methods forinhibiting degradation of a protein in a cell. A method of the inventioncomprises contacting a cell with a polypeptide having an R-X-R sequencein an amount sufficient for inhibiting degradation of a cell surface orsecreted protein. The invention also provides methods for detecting thepresence of an export-incompetent protein in a cell. A method of theinvention comprises contacting a cell with a polypeptide having an R-X-Rsequence and detecting the intracellular transport of the protein. Thevarious assays for detecting transport have been set forth herein, usingan enzymes, such as endoglycosidases, cell surface immunolabeling etc.Such methods are equally applicable in a method for detecting thepresence of an export-incompetent protein in a cell.

In accordance with the present invention, there are provided kits fortreating subjects having or suspected of having a physiological disorderor condition associated with an export-incompetent protein. A kit of theinvention contains one or more AFTs or a polypeptide containing an AFTsequence, and a label or packaging insert for treating a physiologicaldisorder associated with an export-incompetent protein as set forthherein in suitable packaging material. In various aspects, the kitscontain R-X-R tripeptides, preferably polypeptides having an R-X-Rsequence at least 4 amino acids long, more preferably polypeptideshaving an R-X-R sequence at least 5 amino acids long, and mostpreferably polypeptides having R-X-R sequence at least 6 amino acids orlonger. It is understood that any invention polypeptide can be includedin the kits of the invention. Particular embodiments include peptideshaving the sequences GYRQRLE (SEQ ID NO:1), EYRYRSV (SEQ ID NO:2),GQRARIS (SEQ ID NO:3) and QARRRQS (SEQ ID NO:4).

As used herein, the term “packaging material” refers to a physicalstructure housing the components of the kit, such as inventionpolypeptides and nucleic acids. The packaging material preferablymaintains the components sterilely, and can be made of material commonlyused for such purposes (e.g., paper, corrugated fiber, glass, plastic,foil etc.). The label or packaging insert indicates that the kit is tobe used in a method of the invention, for example, for treating CF.

As disclosed herein, the substitution of wild-type CFTR arginine framedpeptide sequence or other signaling motifs in the C-terminal extensiondomain may increase transport of the substituted wild-type CFTR overunsubstituted wild-type CFTR. Thus, an amount of CFTR transported to thecell surface can be increased by such substitutions, even in anexport-competent wild-type CFTR. Accordingly, it is contemplated thatsubstituted polypeptides that utilize the intracellular transportpathway can be produced in greater amounts than unsubstitutedpolypeptides by virtue of this increased transport. For example,secretion of a recombinant polypeptide can be increased by substitutingan arginine framed peptide or one of the C-terminal extension motifs inthe protein.

Thus, in accordance with the present invention, there are providedmethods for increasing the biological production of recombinantpolypeptides. A method of the invention comprises transforming a hostwith a nucleic acid encoding a recombinant polypeptide having asubstituted arginine framed peptide sequence under conditions allowingproduction in an amount greater than unsubstituted polypeptide, therebyincreasing biological production of the recombinant polypeptide.

As used herein, the term “biological production” refers to theproduction of a polypeptide by a living cell, tissue, organ or entireorganism, such as an animal. The methods of the invention for increasingthe biological production of useful polypeptides can be applied tonaturally occurring cell surface, secreted, endosomal or lysosomalpolypeptides. Additionally, the production of polypeptides engineered toutilize the intracellular transport pathway, by attaching anintracellular transport targeting sequence, for example, can similarlybe increased.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims. The invention isfurther described in the following examples, which do not limit thescope of the invention(s) described in the claims.

EXAMPLE I

This example shows that particular signaling motifs mediate orcontribute to the export incompetent characteristic of ΔF508 CFTR.

Inspection of the CFTR amino acid sequence reveals four arginine-framedtripeptide (AFT) sequences, one in the N-terminal cytoplasmic domain,two in the first nucleotide-binding domain (NBD1) and one in theR-domain (FIG. 1A). Because of the sensitivity of CFTR to misprocessingdue to missense mutations in its cytoplasmic domains (Seibert F. S etal. (1995) J. Biol. Chem. 270:2158–2162) the most conservativesubstitutions of these triplets i.e. substitutions of a lysine for thearginine at position 1 or 3, were made (FIG. 1B).

Each of the arginine to lysine substitutions were introduced intoplasmids containing either the wild-type or ΔF508 CFTR sequences.Briefly, a full-length human CFTR cDNA in pNUT vector (Chang, X.-B. etal. (1993). J. Biol. Chem. 268:11304–11311) was utilized as a templatefor site-directed mutagenesis (QuickChange Site Directed MutagenesisKit, Stratagene, La Jolla, Calif.) by polymerase chain reaction (PCR)according to the manufacturer's recommendation. The followingoligonucleotides were used to introduce R29K, R516K, R555K and R766Kinto wild-type CFTR cDNA. R29K:CAATTTTGAGGAAAGGATACAAACAGCGCCTGGAATTGTCAG (SEQ ID NO:10) andCTGACAATTCCAGGCGCTGTTTGTATCCTTTCCTCAAAATTG (SEQ ID NO:11); R516K:CCTATGATGAATATAAATACAGAAGCCTCATC (SEQ ID NO:12) andGATGACGCTTCTGTATTTATATTCATCATAGG (SEQ ID NO:13), R555K:GGAGGTCAACGAGCAAAAATTTCTTTAGCAAGAG (SEQ ID NO:14) andCTCTTGCTAAAGAAATTTTTGCTCGTTGACCTCC (SEQ ID NO:15), R766K:CTTCAGGCACGAAGGAAGCAGTCTCTCCTGAACC (SEQ ID NO:16) andGGTTCAGGACAGACTGCTTCCTTCGTGCTGAAG (SEQ ID NO:17). To combine four lysinesubstitutions in one cDNA, the following fragments were prepared: DraIII-Kpn I fragment which contains part of wild-type CFTR cDNA from nt3328 to 4721 and most of the pNUT expression vector; Kpn I-Afl IIfragment which contains part of the pNUT expression vector and part ofCFTR from nt 72 to 993 covering the R29K mutation; Afl II-Dra IIIfragment from nt 994 to 1777 covering the R516K substitution; DraIII-EcoR I fragment from nt 1778 to 2230 covering the R555Ksubstitution; and EcoR I-Dra III fragment from nt 2231 to 3327 coveringthe R766K substitution. These five fragments were ligated together togenerate full length CFTR cDNA in pNUT (T4 DNA ligase, New EnglandBiolabs, Beverly, Mass.). The sequences of the four fragments coveringthese substitutions were verified after insertion into the pNUT-CFTR. Tocombine the cystic fibrosis causing mutation, ΔF508, with eachindividual or all four of the above substitutions, the same strategy wasutilized, except that pNUT-ΔF508 CFTR was utilized as template.pNUT-ΔF508 CFTR/R29K was made by ligating the following three fragmentstogether, Kpn I-Afl II fragment which contains part of pNUT vector andpart of CFTR from nt 72 to 993 covering the R29K substitution, AflII-Hpa I fragment from nt 994 to 2463 covering the ΔF508 substitution,and Hpa I-Kpn I fragment which contains the part of CFTR from nt 2464 to4721 and part of the pNUT vector. The sequences covering ΔF508, R29K,R516K, R555K and R766K were verified after insertion into the expressionvector pNUT.

Baby hamster kidney cells (BHK) cells grown at 37° C. in 5% CO₂ wereemployed as hosts for CFTR expression as described previously (Loo etal. (1998) EMBO J. 17:6879–6887; and Seibert, F. S et al. (1995) J.Biol. Chem. 270:2158–2162). Subconfluent BHK cells were transformed withthe above wild-type or ΔF508 CFTR constructs using calcium phosphateprecipatation (Chen, C., and Okayama, H. (1987) Mol. Cell. Biol.7:2745–2752). After growth in selective media containing 500 μMmethotrexate, individual colonies were picked and amplified in the sameselective media.

BHK cells were grown, collected and then lysed with 1% SDS; the DNA wassheared by cycling through a 22 gauge needle. Protein lysate (20 μg)from the sample were then subjected to SDS-PAGE on 7% polyacrylamidegels. Protein was electroblotted from the gel to nitrocellulose membraneand the membrane was probed with a mouse anti-CFTR monoclonal antibody,M3A7 (Kartner, N. et al. (1992) Nat. Genetics 1:321–7). Chemiluminescentdetection was performed according to the manufacturer's recommendations(Pierce).

For endoglycosidase H treatment, cell lysates were diluted with 10volumes of buffer (50 mM sodium acetate, pH 5.3, 0.5% Nonidet P-40, 1%β-mercaptoethanol plus a protease inhibitor mixture: 2 μml aprotinin,120 μml benzamidine, 3.5 μl E 64, 1 μg/ml leupepetin and 50 μg/mlaprotinin, 120 μg/ml benzamidine, concentration of 0.1%. Samples werethen incubated for 4 at 37° C. with 10 mU of endoglycosidase H(Boehringer Mannheim). The reactions were stopped by addition of 4volumes of cold ethanol (−20° C.) to precipitate the proteins.Precipitated proteins were then resuspended in gel loading dye andsubjected to SDS-PAGE and immunoblotting as above.

Western blots of BHK cell extracts revealed that these four R→Ksubstitutions individually or in combination did not dramatically changethe steady state amounts of mature, fully glycosylated band (˜170 kDa)or immature core-glycosylated band (˜150 kDa) of wild-type CFTR (FIG.1C, lanes 1–6). Thus, synthesis and processing was not impaired by anyof these mutations. In the case of ΔF508 CFTR, however striking changeswere observed. Without any additional mutation, it displays the usualdoublet band pattern with the same major core-glycosylated band (˜150kDa) as the wild-type plus a smaller band at ˜135 kDa (lane 7). Thelatter is missing an N-terminal fragment of ˜15 kDa due to alternativeinitiation of translation or a specific proteolytic clip and is muchmore prominent in mutants such as ΔF508 than in wild-type CFTR This bandremains prominent in 3 of the 4 R→K variants (R516K, R555K, R766K; lanes9, 10, and 11 respectively) but not in R29K (lane 8) or in “4RK (lane12).” More significantly, however, bands of lower mobility are producedby the R29K and 4RK variants (lanes 8 and 12). These are not identicalin size but they appear to be at either edge of the broad band displayedby wild-type CFTR because of heterogeneous N-glycosylation. The relativeamount is greater in the 4RK combination mutant.

Endoglycosidase-H insensitivity of these large bands formed by the R29Kand 4RK versions of ΔF508 CFTR confirms that indeed theiroligosaccharide chains have been extended by Golgi glycosyl transferases(FIG. 1D, arrowhead). Further evidence that these bands were present atthe cell surface came from their accessibility to an impermeant aminoreactive biotinylation reagent added to intact cell monolayers.Therefore, substitution of one of the obligatory arginines, in the RQRtriplet is able to partially overcome the maturation arrest of ΔF508;this effect is much more pronounced when similar substitutions are madein all four triplets.

For cell surface biotinylation studies, cells grown to about 90%confluency were treated with 2 mM sodium butyrate for 18 h to augmentCFTR expression. Dishes of cells were then placed on ice in a cold roomfor 30 minutes and all subsequent steps were performed. Cells washedwith PBS containing 0.1 mM CaCl₂ and 1.0 mM MgCl₂, were labeled using 3ml of 1 mg/ml EZ-Link Sulfo-NHS-SS-biotin (Pierce) in PBS, pH 8.0 for 30minutes. Following washes in PBS+1% BSA and PBS, cells were incubatedfor 30 minutes in NP40 lysis buffer (0.09% NP40, 150 mM NaCl, 10 mMNH₄MoO₄, 50 mM Tris-HCl, pH 7.4), scraped and transferred to microfugetubes. Insoluble materials were removed by centrifugation. Biotinylatedproteins were then precipitated by incubation overnight with 50 μl ofpacked immobilized streptavidin beads (Pierce). Beads were washed withRIPA buffer (0.1% SDS, 1% DOC, 1% Triton X-100, 150 mM NaCl, 50 mMTris-Hcl, pH 7.4) and proteins eluted with gel sample buffer forelectrophoresis and Western blotting with M3A7 as detailed above.

The results in FIG. 1D show that the mature band of substituted ΔF508CFTR (ΔF508/4RK) was accessible to the impermeant amino reactivebiotinylation reagent added to the intact cell monolayers. These resultsconfirm that substitution of AFTs can overcome the biosynthetic arrestof ΔF508 CFTR

EXAMPLE II

This example shows that export of substituted wild-type and ΔF508 CFTRfrom the endoplasmic reticulum and subsequent maturation is kineticallyincreased in comparison to unsubstituted wild-type and ΔF508 CFTR.

Pulse chase studies were performed to determine the rate at whichmaturation occurred in the arginine-framed triplet substituted wild-typeand ΔF508 CFTR polypeptides (FIG. 2). Briefly, cells expressingunsubstituted and substituted wild type or ΔF508 CFTR were starved for30 minutes by incubation in methionine-free medium and then labeled for20 minutes with 100 μCi/ml of ³⁵S-methionine (>800 Ci/mmol; Amersham).Cells were lysed at the end of the labeling period (0 chase time) orwere further incubated in complete medium containing 5% fetal bovineserum and 1 mM methionine for the indicated times (chase). Cells werecollected and lysed with the NP40 lysis buffer described above andcentrifuged at 15,000×g for 15 min at 4° C. to obtain a solublesupernatant which was incubated overnight with the M3A7 primaryantibody. Protein G-agarose (Gibco-BRL) was then used to remove thecomplexes formed which were washed four times with RIPA buffer,dissolved in electrophoresis sample buffer, and subsequentlyfractionated on SDS-7% acrylamide gels. Following fractionation, thegels were fixed in 30% methanol and 10% acetic acid, equilibrated in 1Msodium salicylate and dried for fluorography. Electronic autoradiographywas performed with a Packard Instant Imager. Conversion of³⁵S-methionine pulse labeled precursor form of CFTR (time 0) to matureproduct after chase times (1, 2, 3 and 5 hour) are indicated.

At the end of the 30 min pulse (0 min), maturation of the wild-type CFTRprecursor substituted with four lysine substitutions (wild-type/4RK) wasalready evident. In contrast, no maturation for unsubstituted wild-typeCFTR was observed at the end of the pulse (compare FIGS. 2A to 2B).

Conversion of the substituted ΔF508/4RK CFTR precursor to mature endoHresistant oligosaccharide chains is readily apparent whereas with ΔF508CFTR, there is no conversion of the core-glycosylated precursor to anyhigher molecular weight product (compare FIGS. 2C to 2D). Electronicautoradiography of the same dried gels indicated that approximately 10%of the radioactivity in the pulse-labeled immature ΔF508/4RK band (0time) was present in the larger mature band after 4 h of chase (FIG.2D). This compares with ˜30% conversion of precursor to product in thecase of wild-type CFTR. Hence, the extent of maturation of ΔF508/4RKviewed in this way is generally similar to the steady-state proportionreflected in the immunoblots (FIG. 1C). Furthermore, substitution of allfour AFTs markedly decreased degradation and promoted maturation ofΔF508.

These results indicate that the rate of ER export of export-competentand export-incompetent polypeptides such as CFTR can be increased bysubstitution of AFT sequences as set forth herein.

EXAMPLE III

This example shows that increased maturation of substituted wild-typeand ΔF508 CFTR can be observed directly in cells.

Immunofluorescence microscopy in cells expressing ΔF508 CFTR wasperformed using the highly specific monoclonal antibody, M3A7 (Kartneret al., 1992, supra). Briefly, transformed cells were grown oncoverslips, fixed in 70% cold methanol at −20° C. for 10 minutes,blocked using 1% BSA and 5% normal rabbit serum in PBS and werepermeabilized by incubating in PBS+0.1% saponin for 1 h at 4° C. CFTRwas detected by incubation with 10 μg/ml of M3A7 primary antibody for 60min at room temperature. Rhodamine linked rabbit anti-mouse secondaryantibody (DAKO) was diluted 1:50 in the same blocking solution for afurther 1 h incubation. Photomicrographs were made using a DiagnosticInstruments Spot Cam digital camera on a Nikon Microphot-FXA microscope.

The results in FIG. 3 indicate that four R→K substitutions of wild-typeCFTR does not alter its normal distribution, with perinuclear stainingindicating ER localization of the immature CFTR in addition to stronguniform staining of mature CFTR over the entire cell surface (FIGS. 3Aand 3B). Perinuclear staining only was observed in cells expressingΔF508 CFTR indicating ER retention (FIG. 3C). In contrast, there isextension of the staining more peripherally in cells expressingΔF508/4RK CFTR (FIG. 3D). Although there is still less peripheralstaining for ΔF508RK CFTR than for wild-type CFTR, this is expectedbased upon the relative amounts of the mature CFTR with complexoligosaccharide chains (FIG. 1C and FIG. 2).

EXAMPLE IV

This example shows that polypeptides containing an AFT sequence caninduce or enhance transport of export incompetent ΔF508 CFTR from theendoplasmic reticulum to the cell surface.

Heptamer peptides including two residues from the CFTR sequence oneither side of the AFT were synthesized (Molecular Biology Core of theMayo Clinic, Rochester, Minn.). BHK cells expressing either wild-type orΔF508 CFTR-GFP fusion protein (Loo et al., 1998, supra) were allowed totake up a mixture of the heptamers by scrape loading Malcolm et al.,(1996) J. Biol. Chem. 271:13135–9) and plated on cover slips forvisualization the next day using a fluorescence microscope with an FITCfilter. Localization of wild-type CFTR-GFP fusion with or withoutexposure to heptamers was the same as antibody staining of wild-typeCFTR. Cells expressing the ΔF508 CFTR-GFP fusion protein in the absenceof heptamers also exhibited the same perinuclear clustering as wasobserved with antibody staining of unfused ΔF508 CFTR. In contrast,heptamer treatment of cells expressing the ΔF508 CFTR-GFP fusion proteinexhibited more peripheral fluorescence staining.

The results in FIG. 4 show that an AFT sequence can competitivelyinhibit recognition of AFT containing nascent polypeptides therebyinhibiting ER retention and promoting transport of polypeptidescontaining AFT sequence from the endoplasmic reticulum.

EXAMPLE V

This example shows that the R→K substitutions result in a functionallyactive ΔF508 CFTR at the cell surface.

Wild-type and ΔF508 CFTR expressing cells with each of the individualR□K substitutions, or all four lysine substitutions were grown toconfluence in six well culture dishes. The cells were washed with amodified Ringer's buffer solution (136 mM NaNO₃, 3 mM KNO₃, 2 mMCa(NO₃)₂, 2 mM Mg(NO₃)₂, 10 mM glucose and 20 mM HEPES, pH 7.4) andloaded for 60 min at room temperature with the same buffer supplementedwith 1 μCi Na³⁶Cl (Amersham) in a volume of 0.5 ml per well. Wells werethen washed three times at one min intervals. Samples (0.5 ml) were thencollected at 1 minute intervals for scintillation counting withstimulation occurring at time 0 by adding buffer containing 1 mM IBMX,10 μM Forskolin and 100 μM dibutyryl cyclic AMP. The 0.5 ml samples werecollected into 24 well Top Count plates (Packard) and 1 ml of Microcint40 scintillation cocktail (Packard) was added. ³⁶Cl⁻ radioactivity wasdetermined in a Packard Top Count scintillation counter.

The influence of each of the R→K substitutions on the cAMP stimulatedefflux of ³⁶Cl⁻ from wild-type or ΔF508 CFTR expressing cells loadedwith ³⁶Cl⁻ anion is shown in FIG. 5A. Within 3 minutes of cAMPstimulation there was a maximal rate of efflux from all cells expressingwild-type CFTR with or without any or all of the R→K mutants. Nodetectable increase in efflux rate from cells expressing ΔF508 CFTRalone or with the R516K, R555K or R766K substitution was observed. Incells expressing ΔF508/R29K, low rates of ³⁶Cl⁻ efflux occurred atdelayed times after stimulation. In cells expressing ΔF508 CFTR combinedwith all four R→K substitutions (4RK) the maximal rates of effluxobserved were more than half as rapid as those from cells expressingwild-type CFTR, confirming that mature ΔF508/4RK CFTR does function atthe cell surface.

To further characterize the Cl⁻ channel activity, membrane vesicles wereisolated from cells expressing wild-type, wild-type/4RK and ΔF508/4RKCFTR, fused with planar lipid bilayers and single channels were recorded(Aleksandrov, A. A., and Riordan, J. R. (1998) FEBS Lett. 431:97–101).Vesicles were prepared and phosphorylated with PKA as describedpreviously (Aleksandrov and Riordan, 1998, supra). Briefly, planar lipidbilayers were painted onto a 0.2 mm hole drilled in a Teflon cup using aphospholipid solution in n-decane containing a 2:1 mixture of1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (20 mg/ml). The lipidbilayer separated 1.0 ml of solution in the Teflon cup (cis-side) from4.0 ml of solution in an outer glass chamber (trans-side). Both chamberswere magnetically stirred and thermostated. Heating and temperaturecontrol were established using a Temperature Control System TC2BIP (CellMicroControls). Electrical contact with the solutions was provided byAg/AgCl electrodes through agar bridges filled with 0.5 M KCl. Themembrane potential difference was measured as the difference betweentrans and cis side potentials. The trans side was grounded andelectrical measurements of the single channel current were performedunder voltage clamp conditions using an Axopatch 200B (Axon Instruments)amplifier. The output signal was filtered with an 8-pole low pass BesselFilter LPBF-48DG (NPI Electronic GmbH) with cut off frequency of 50 Hzand recorded on magnetic tape using a VR-10B (Instrutech Corp.) digitaldata recorded on a Sony SLV-440 VCR. For data analysis, the signal wasdigitized (Digidata 1200; Axon Instruments) with a sampling rate of 500Hz and analyzed using pCLAMP 6.0 (Axon Instruments) software. Origin 4.0(Microcal) software was used to fit all points histograms by multi-peakGaussians. Membrane vesicles were prephosphorylated with PKA and addedas a concentrated stock solution to the cis side to obtain a finalprotein concentration of 10–15 μg/ml. Measurements were made insymmetrical salts solutions containing, in mM: 300 Tris-HCl, 2 MgCl, 1EGTA, pH 7.2, at a membrane potential of −75 mV. MgATP (2 mM) was addedto the cis side only. Under these conditions any cationic channelspresent in the membrane vesicles are invisible and CFTR chloridechannels appeared only after fusion of vesicles with an inside outorientation. Conductances for wild-type, wild-type 4RK and ΔF508/4RKwere 10.7, 10.8, and 10.7 pS, respectively. Mean open times τ₀ were 230,1400, and 700 msec respectively. Short mean closed times τ_(c1) were 10msec in each case. Long mean closed times τ_(c2) were 680, 2000, and2800 msec, respectively. Conductance as a function of Cl⁻ concentrationand temperature as well as anion selectivity of the CFTR ion channelwere tested in control experiments showing good agreement with publisheddata (Tabcharani, J. A et al. (1997) J. Gen. Physiol. 1110:341–54).

Compared with wild-type single channels which exhibit an openprobability of ˜0.25 at 25° C., channels generated by wild-type/4RK hada higher mean open probability of −0.61 (FIG. 5B). Thus although thegating kinetics are somewhat altered with both mean open and mean closedtimes increased, the four R→K substitutions certainly do not impedecurrent flow through the channel, consistent with the undiminished rateof ion flux (FIG. 5A). Significantly, the ΔF508/4RK channels had an openprobability similar to wild-type CFTR, confirming the activity revealedby the macroscopic flux measurements. No tracing is shown for ΔF508 CFTRalone as we have never been able to detect CFTR-like single channelsusing vesicles from BHK cells expressing this variant. We conclude thatthe channels observed from ΔF508/4RK CFTR are entirely due to thematuration that is enabled by the release from ER retention when theAFTs are substituted.

The above examples show that substitution of one of the arginines ineach of the four AFTs, individually or simultaneously, permits nascentΔF508 CFTR to mature about one-third as efficiently as wild-type CFTRand generates functional chloride channels at the cell surface. Thus,release of ΔF508 CFTR from ER retention by interfering with recognitionof these tripeptide signals may provide the basis of a novel therapeuticstrategy for cystic fibrosis.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated polypeptide selected from the group consisting of:GYRQRLE (SEQ ID NO:1, EYRYRSV (SEQ ID NO:2), GQRARIS (SEQ ID NO:3) andQARRRQS (SEQ ID NO:4).
 2. An isolated polypeptide having one or moreR-X-R sequences, wherein at least one R of an R-X-R sequence has beensubstituted with another amino acid, and wherein the substitutedpolypeptide is exported from the ER in an amount or at a rate greaterthan the unsubstituted polypeptide wherein the rate is measured in thesame cell, and wherein said polypeptide is CFTR Cystic FibrosisTransmembrane Regulator (CFTR).
 3. A formulation consisting essentiallyof an isolated polypeptide consisting of GYRQRLE (SEQ ID NO:1), EYRYRSV(SEQ ID NO:2), GQRARIS (SEQ ID NO:3), or QARRRQS (SEQ ID NO:4), whereinsaid formulation is in a pharmaceutically acceptable carrier.